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Mastermind is a putative activator for Notch

2000; Elsevier BV; Volume: 10; Issue: 13 Linguagem: Inglês

10.1016/s0960-9822(00)00577-7

1879-0445

Andrei G. Petcherski, Judith Kimble,

Tissue Engineering and Regenerative Medicine

During signaling by the Notch receptor, Notch's intracellular domain is cleaved, moves to the nucleus and associates with a DNA-binding protein of the CSL class (CSL for CBF1, Suppressor of Hairless (Su(H)), LAG-1); as a result, target genes are transcriptionally activated (reviewed in [1Artavanis-Tsakonas S Rand M.D Lake R.J Notch signaling cell fate control and signal integration in development.Science. 1999; 284: 770-776Crossref PubMed Scopus (4675) Google Scholar, 2Artavanis-Tsakonas S Matsuno K Fortini M.E Notch signaling.Science. 1995; 268: 225-232Crossref PubMed Scopus (1365) Google Scholar]). In Caenorhabditiselegans, a glutamine-rich protein called LAG-3 forms a ternary complex with the Notch intracellular domain and LAG-1 and appears to serve as a transcriptional activator that is critical for signaling [3Petcherski A.G Kimble J LAG-3 is a putative transcriptional activator in the C. elegans Notch pathway.Nature. 2000; 405: 364-368Crossref PubMed Scopus (152) Google Scholar]. Although database searches failed to identify a LAG-3-related protein, we surmised that Notch signaling in other organisms might involve an analogous activity. To search for a LAG-3-like activity in mice, we used a modified yeast two-hybrid screen similar to that used to identify LAG-3 [3Petcherski A.G Kimble J LAG-3 is a putative transcriptional activator in the C. elegans Notch pathway.Nature. 2000; 405: 364-368Crossref PubMed Scopus (152) Google Scholar]. Briefly, we used a complex bait to screen a library of mouse cDNAs fused to the Gal4 activation domain (Clontech). That bait included mouse CBF1 fused to the Gal4 DNA-binding domain (GD) as well as the intracellular domain of mouse Notch1. The bait proteins were co-expressed from a pBridge vector. Out of 6 million transformants, we recovered one positive with similarity to Drosophila Mastermind and human KIAA0200 (Figure 1a). We focused on this clone because Drosophila Mastermind is known to be critical for Notch signaling (reviewed in [2Artavanis-Tsakonas S Matsuno K Fortini M.E Notch signaling.Science. 1995; 268: 225-232Crossref PubMed Scopus (1365) Google Scholar]) [4Lehmann R Jimenez F Dietrich U Campos-Ortega J On the phenotype and development of mutants of early neurogenesis in Drosophila melanogaster.Wilhelm Roux's Archiv Dev Biol. 1983; 192: 62-74Crossref Scopus (354) Google Scholar, 5Xu T Rebay I Fleming R.J Scottgale T.N Artavanis-Tsakonas S The Notch locus and the genetic circuitry involved in early Drosophila neurogenesis.Genes Dev. 1990; 4: 464-475Crossref PubMed Scopus (109) Google Scholar]. We call the murine ortholog of Mastermind mMam1, and the human one hMam1. The mMam1 fragment recovered in the two-hybrid screen consisted of 62 amino acids and included a conserved region present in both fly and human Mastermind proteins (Figure 1). To explore the idea that Mastermind might have a role similar to LAG-3 in Notch signaling, we conducted a series of two-hybrid assays (Figure 2). We first showed that mMam1 bound mCBF1–GD in the presence of either Notch1 or Notch3, but not in their absence (Figure 2a). We next asked whether Drosophila Mastermind might participate in a similar complex in flies. We made a fusion protein carrying the Gal4 activation domain and the amino-terminal 198 amino acids of fly Mastermind (dMam (1–198), Figure 1a; henceforth called dMam), which includes the conserved region of Mastermind that is critical for complex formation among mouse components. We found that dMam bound Su(H) strongly in the presence of the fly Notch intracellular domain, but not in its absence (Figure 2b). We next explored the interchangeability of proteins from different species. Remarkably, the fly protein, dMam, interacted with murine Notch1 or Notch3 and murine CBF1 (Figure 2c), and mMam1 interacted with fly Notch and Su(H) (Figure 2d). In contrast, C. elegans LAG-3 did not form a complex with either murine or fly components (Figure 2e), and mMam and dMam did not complex with worm components (Figure 2f). We conclude that both fly and murine Mastermind proteins form a ternary complex with either fly or murine receptors and CSL proteins. This interchangeability underscores the similarity between the fly and murine Notch pathways. Although murine Mastermind is not described, a full-length cDNA sequence for human Mastermind is available. Comparison of human and fly Mastermind sequences reveals only one short region of significant similarity that is limited to 60 amino acids at the amino terminus (Figure 1). Therefore, despite a low overall sequence similarity between mouse and Drosophila Mastermind proteins, the region crucial for complex formation is conserved. Finally, we examined the importance of the receptor's ankyrin repeats for complex formation. In C. elegans, formation of the ternary complex is dependent on the ankyrin repeats of the Notch-related receptor GLP-1 [3Petcherski A.G Kimble J LAG-3 is a putative transcriptional activator in the C. elegans Notch pathway.Nature. 2000; 405: 364-368Crossref PubMed Scopus (152) Google Scholar]. To ask whether the same situation holds for the murine complex, we used two missense mutants, M1 and M2, each of which bears amino-acid substitutions in the fourth ankyrin repeat of mNotch1 [6Kopan R Nye J.S Weintraub H The intracellular domain of mouse Notch a constitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD.Development. 1994; 120: 2385-2396Crossref PubMed Google Scholar]. Consistent with results in C. elegans, both M1 and M2 compromised interactions among Notch1, CBF1 and either mMam1 or dMam (Figure 2). What is the role of Mastermind in Notch signaling? Previous studies suggested a role in transcriptional control. In Drosophila, Mastermind is a nuclear protein [7Smoller D Friedel C Schmid A Bettler D Lam L Yedvobnick B The Drosophila neurogenic locus mastermind encodes a nuclear protein unusually rich in amino acid homopolymers.Genes Dev. 1990; 4: 1688-1700Crossref PubMed Scopus (109) Google Scholar] and is bound to chromatin [8Bettler D Pearson S Yedvobnick B The nuclear protein encoded by the Drosophila neurogenic gene mastermind is widely expressed and associates with specific chromosomal regions.Genetics. 1996; 143: 859-875PubMed Google Scholar]. Furthermore, in Drosophila, Mastermind acts downstream of Notch in signaling [9Schuldt A.J Brand A.H Mastermind acts downstream of Notch to specify neuronal cell fates in the Drosophila central nervous system.Dev Biol. 1999; 205: 287-295Crossref PubMed Scopus (30) Google Scholar]. The amino-acid sequences of both human and fly Mastermind proteins are rich in glutamine and proline (see below), a common feature in transcriptional activators [10Mitchell P.J Tjian R Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins.Science. 1989; 245: 371-378Crossref PubMed Scopus (2152) Google Scholar]. In the work reported here, we provide a physical link between Mastermind and the major CSL transcription factor of the Notch pathway. We also show that the interaction of both mMam and dMam with the Notch intracellular domain and CBF1 relies on the receptor's ankyrin repeats (Figure 2g). These repeats are essential for Notch signaling and the transcriptional response. In C. elegans, point mutations in the ankyrin repeats severely compromise signaling by the Notch-related receptor GLP-1 [11Kodoyianni V Maine E.M Kimble J Molecular basis of loss-of-function mutations in the glp-1 gene of Caenorhabditis elegans.Mol Biol Cell. 1992; 3: 1199-1213Crossref PubMed Scopus (104) Google Scholar]. In tissue culture cells, the M1 and M2 point mutations abolish receptor function [6Kopan R Nye J.S Weintraub H The intracellular domain of mouse Notch a constitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD.Development. 1994; 120: 2385-2396Crossref PubMed Google Scholar] and compromise the activation of transcription by Notch signaling [12Jarriault S Brou C Logeat F Schroeter E.H Kopan R Israël A Signaling downstream of activated mammalian Notch.Nature. 1995; 377: 355-358Crossref PubMed Scopus (1195) Google Scholar, 13Kato H Taniguchi Y Kurooka H Minoguchi S Sakai T Nomura-Okazaki S Tamura K Honjo T Involvement of RBP-J in biological functions of mouse Notch1 and its derivatives.Development. 1997; 124: 4133-4141Crossref PubMed Google Scholar]. The simplest explanation for all these findings is that Mastermind functions as a transcriptional activator for Notch signaling. We note important parallels between LAG-3 in C. elegans and Mastermind in Drosophila and mammals. First, all of these proteins form a ternary complex with an intracellular fragment of Notch and a CSL DNA-binding protein. Second, mutations in the fourth ankyrin repeat of the receptor compromise ternary complex formation for C. elegans[3Petcherski A.G Kimble J LAG-3 is a putative transcriptional activator in the C. elegans Notch pathway.Nature. 2000; 405: 364-368Crossref PubMed Scopus (152) Google Scholar] and mouse proteins, as we report here. Third, all three proteins are rich in glutamine and proline: 27.6% in LAG-3, 29.4% in dMam and 22% in hMam1. Fourth, LAG-3 and Mastermind function downstream of Notch in C. elegans[3Petcherski A.G Kimble J LAG-3 is a putative transcriptional activator in the C. elegans Notch pathway.Nature. 2000; 405: 364-368Crossref PubMed Scopus (152) Google Scholar] and Drosophila[9Schuldt A.J Brand A.H Mastermind acts downstream of Notch to specify neuronal cell fates in the Drosophila central nervous system.Dev Biol. 1999; 205: 287-295Crossref PubMed Scopus (30) Google Scholar], respectively. We propose that LAG-3 and Mastermind perform analogous functions as activators for Notch. What is the evolutionary relationship between LAG-3 and Mastermind? An intriguing idea is that LAG-3 and Mastermind share a common ancestor. The conservation in amino-acid sequence between Mastermind orthologs is much lower than is found for other components of the pathway: whereas hMam1 and dMam share similarity only in a stretch of 60 amino acids within a much larger protein (Figure 1), Notch and CSL proteins show high similarity (44.8% and 74.5% identity for hNotch1/dNotch and hCBF1/Su(H), respectively) over most of their length between these same species. It therefore seems plausible that the absence of similarity between LAG-3 and Mastermind may reflect a high rate of amino-acid substitution in these proteins rather than a distinct evolutionary origin. We thank Rafi Kopan and Meera Saxena for providing mNotch1 plasmids. AGP is an HHMI predoctoral fellow. JK is an investigator of the Howard Hughes Medical Institute.